The interaction of the bacteria on the tooth surface, the dental plaque or oral biofilm, the diet, and the teeth acting together over time all contribute to dental caries. Fundamentally, an imbalance between demineralization and remineralization is what causes tooth decay.^[1]

Remineralization is the net gain in minerals that were previously lost due to demineralization at the surface of the enamel. Early treatment of developing carious lesions seeks to remineralize a subsurface lesion that is still active but not cavitating, preventing further mineral loss from cavitating the lesion.^[1]

In the past few years, dentists have employed a variety of topical agents such as gels and varnishes, to treat WSLs. Subsurface caries tend to remineralize with these substances by giving out calcium phosphate, either with or without fluoride, and managing the microenvironment.^[1]

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), nano-hydroxyapatite (nHAp), and erbium-doped yttrium aluminum garnet (Er:YAG) laser have been studied for remineralization of incipient caries in permanent teeth. However, not many studies have been done to analyze the action of Er:YAG laser in combination with nHAp and CPP-ACP for primary enamel remineralization. Such research will aid in employing lasers to improve the action of remineralizing agents. It may reduce the number of applications of such agents and the overall number of dental visits for preventive and remineralization therapy.

Hence, this study aimed to evaluate the effect of nHAp and CPP-ACP with and without Er:YAG laser irradiation on the microhardness and surface morphology of demineralized primary enamel.

The study was conducted on primary incisor teeth collected from the Department of Pediatric and Preventive Dentistry, Bharati Vidyapeeth (Deemed to be University), Dental College and Hospital, Sangli. The study performed was an experimental in vitro study.

Table 1 shows the comparison of microhardness at baseline.

The results revealed that there was a nonsignificant difference in microhardness of the four groups at baseline.

Table 2 shows the comparison of microhardness before and after demineralization in each group.

There was a significant reduction in the microhardness of each group after demineralization.

Table 3 shows the comparison of microhardness before and after remineralization in each group.

The results of Table 3 and Graph 1 showed that there was a nonsignificant difference in the microhardness of the four groups after remineralization.

Table 4 shows the comparison of the percentage increase in the microhardness of four groups after remineralization.

According to Table 4 and Graph 2, the highest increase was seen in Group 4 followed by Group 3 and the least in Group 1. However, there was a nonsignificant difference in the percentage increase in the microhardness of the four groups after remineralization.

SEM images revealed the following findings:

For sound enamel surface [Figure 5a], normal, smooth, and intact enamel surface structure devoid of any alteration was noted before demineralization; normal perikymata appeared to run in parallel lines. Some porosities could be seen.

The structure of the enamel surface after demineralization is changed in Figure 5b. Enamel lost its normal architecture and the prisms showed irregularity. Numerous voids and microporosities were seen on the enamel surface.

In Figure 5c, the changes in enamel surface structure after treatment with CPP-ACP (Group 1) were demonstrated. Morphological changes were observed by the presence of amorphous, globular, and crystalline structures that occlude the micropores created after demineralization.

In Figure 5d, most of the microporosities were hidden and occluded. The nHAp particles (Group 2) appeared to be melted, recrystallized, and trapped inside the pores forming a plug.

Figure 5e and f shows the SEM image of the enamel surface irradiated with Er:YAG (Group 3, 4) laser. It showed an absence of normal enamel perikymata, cracks, craters, etc. Irregular melted and recrystallized areas were seen on the enamel surface.

The SEM image of the enamel surface treated with Er:YAG laser + CPP-ACP (Group 3) in Figure 5e shows that the interprismatic substances were evident, with porosities and areas of remineralization. It also displayed thick and more frequent lines of remineralization along the prismatic borders; certain areas of calcifications were evident along the porosities.

The SEM image of the enamel surface treated with Er:YAG laser + nHAp (Group 4) in Figure 5f showed that the surface defects that were produced by laser irradiation were reconstructed by the deposition of a layer of nHAp particles.

The enamel must endure a variety of physical and chemical processes, such as compressive stresses (up to around 700N), physiologic and pathologic wear of tooth structure, and most critically, an acidic environment brought on by the presence of bacterial plaque and food. Due to their proximity to saliva and salivary plaque, the HAp crystals near the enamel’s surface are always active. The process of demineralization takes place at pH 5.5, where HAp dissolves as a result of acid generated by bacterial metabolic products or by intake of acidic foods.^[5]

If allowed to continue for a long time, significant mineral loss results in enamel loss and subsequent cavitation. Deposition of calcium, phosphate, and fluoride ions (fluorapatite) improves resistance to dissolution by organic acids as fluorapatite crystals have an advancing growth pattern, leading to the formation of large crystals with hexagonal outlines. The demineralization process continues until the pH rises, causing remineralization to occur. Therefore, improving the remineralizing process with the use of remineralization products is the greatest technique for caries control.

The early demineralization of the enamel at the surface and subsurface is known as a WSL, which manifests as a milky white opacity. If left untreated, subsurface demineralization increases porosity, which eventually alters the optical characteristics of the teeth and causes cavitation.^[5]

The use of fluoride dentifrice to regularly brush teeth has been advised for years, however using fluoride continuously at higher than optimal doses has been linked to fluorosis and fluoride poisoning in children, especially those under the age of six. Additionally, a layer that resists acid formation may develop, which hinders the diffusion of remineralizing ions into deeper layers and restricts overall remineralization of the region.^[6]

A potential alternative remineralizing agent with anti-caries properties has been identified based on calcium phosphate. It is CPP-ACP. In low-pH environments, it has been demonstrated to prevent demineralization by releasing calcium and phosphate ions.^[7]

As a result of stabilizing calcium phosphate in solution by binding ACP to numerous phosphoserine residues and forming small clusters of CPP-ACP, the anticariogenic effects of CPP-ACP are related to increasing the buffering effect of saliva, which suppresses demineralization and activates remineralization, or both.^[7]

A biocompatible synthetic substance called nHAp resembles the hydroxyapatite crystals found in human teeth. Its application in numerous curative, reparative, and regenerative therapies has drawn a lot of attention recently.

HA crystals with a size range of 20–40 nm make up the majority of the material in tooth enamel. Due to the structural and chemical similarities between nano-sized HA crystals and enamel apatite crystals, synthetic nHAp is used for remineralization applications. Additionally, nHAp has been demonstrated to have a remineralizing impact on artificial carious lesions and develop a new enamel layer. It is more biocompatible than HA and has stronger bioactivity and mechanical characteristics.^[8]

Numerous studies conducted over the past four decades have demonstrated the use of laser irradiation on dental tissues in preventing enamel erosion or WSLs. When teeth are exposed to the laser, the light and the microscopic components of the dental hard tissue interact. The irradiation energy changes into heat instantly when the particular components absorb the light. Enamel is thought to undergo microstructural and chemical changes as a result of heat, which also accounts for its greater acid resistance.^[7]

In the current investigation, nail polish was applied to the primary incisors’ labial surface, leaving a 2 * 2 mm window. The teeth were sectioned 1 mm below the CEJ and embedded in self-cure acrylic to form tooth models. Each enamel specimen underwent a 48-h demineralization simulation in a 50 ml demineralizing solution made up of 2.2 Mm CaCl2.2H2O, 2.2 Mm NaHPO4.7H2O, and 0.05 M Lactic acid at 37°C in an incubator. This created artificial carious lesions on the enamel samples. All of the samples were tested for postdemineralization microhardness using Vickers testers after being rinsed with distilled water and dried.

In a study by Nair et al. in 2016, the specimens were individually submerged in 5 ml of an acetate buffer solution (0.1 mol/L, pH 4.5) and incubated at 37°C for 24 h to mimic the oral environment.^[9] A hundred specimens were prepared from 50 human premolars to explore the caries-inhibiting effects of remineralizing agents and laser on enamel using an atomic emission spectrometry analysis. The enamel samples were divided into 6 groups at random, including Untreated (Control), CPP-ACP (GC Tooth Mousse), CPPACFP (GC Tooth Mousse Plus), Er:YAG laser therapy alone, CPP-ACP with Er:YAG laser, and CPP-ACFP with Er:YAG laser. They came to the conclusion that, as compared to other groups, the combination of CPP-ACFP and Er:YAG laser was more effective at reducing enamel demineralization.

In the present study, the specimens of Group 1 were treated with CPP-ACP paste. The paste was applied on the surface of the specimen using a disposable micro applicator tip and kept for 3 min, followed by pH cycling. In a study by Oshiro and Yamaguchi, two sets of specimens were placed in 10-fold diluted CPP-ACP paste (Tooth Mousse, GC Corp. Tokyo, Japan) or placebo paste (without CPP-ACP) for 10 min each before being placed in a demineralizing solution.^[10] In a vacuum evaporator, a small layer of gold was applied to the surfaces, and they were then examined using field emission SEM. CPP-ACP paste treatment caused minor morphological alterations in enamel and dentin specimens. It was concluded that CPP-ACP paste may prevent the demineralization of the tooth structure, according to the morphological observations of the enamel and dentin surfaces. The results of the present study demonstrated a significant increase (<0.001) in the microhardness of Group 1 after remineralization (265.85) in comparison to postdemineralization values (181.76).

In group 2, the specimens were treated with nHAp paste. The paste was applied on the surface of the specimen using a disposable micro applicator tip and kept for 3 min, followed by pH cycling. The impact of nHAp and CPP-ACP fluoride paste on artificial enamel carious lesions of young permanent teeth was compared by Abdelaziz et al. in 2019.^[11] For the investigation, 60 extracted premolar teeth were chosen. The enamel surface microhardness was assessed at baseline, after the formation of an enamel lesion, and following therapy. They came to the conclusion that both CPP-ACP fluoride and nHAp were successful at remineralizing young permanent teeth with early caries-like lesions. The present study showed similar findings, where postdemineralization values of Group 3 specimens (176.12) showed a significant increase (<0.001) in the postremineralization values (258.59).

For the specimen in Group 3, the CPP-ACP paste was applied after laser irradiation. The Er: YAG LASER emitting at a wavelength of 2.94 micrometers was used to irradiate the exposed enamel. The parameters were: 80 mJ of energy and a 4 Hz frequency for 10 s and energy per pulse of 80 mJ with water spray and 1.2 mm spot size. The results of the present study showed a significant (<0.001) rise in the microhardness values of this group after remineralization.

For their investigation, Khamverdi et al. used thirty sound maxillary extracted premolars.^[12] The crowns were divided into labial and palatal portions at the cervical line. Specimens were mounted in acrylic blocks. The samples underwent pH cycling before being randomly divided into 4 groups (n = 15), as follows: CG stands for the control group, LAS for CO2, CP for CPP-ACP, and LASCP for laser-combined CPP-ACP therapy. The demineralized enamel surface was treated with CPP-ACP paste for 5 min, followed by washing. The surface of the specimen was irradiated with a CO2 laser at a wavelength of 10.6 μm for LAS and LASCP. Following were the laser’s settings: power of 0.7 W, 50 Hz pulse frequency, 0.2 mm focal spot, 0.4 msond pulse length, noncontact mode, 10 mm between hollow tube tip and tooth surface, and spot size: 0.4 mm. The Vickers microhardness of each sample was determined. According to their findings, the CO2 laser and CCP-ACP were useful for increasing enamel hardness following demineralization. The application of CCP-ACP paste and CO₂ laser irradiation increased the demineralized enamel’s ability to remineralize.

In the present study, for the specimen of Group 4 nHAp paste was applied after laser irradiation. The results of the present study demonstrated a significant increase (<0.001) in the microhardness of Group 4 after remineralization (269.72) in comparison to postdemineralization values (175.08).

Assal et al., prepared pure hydroxyapatite (nHA) and fluoro hydroxyapatite (nFHA), two different forms of nHAp.^[2] They used sixty extracted premolar teeth which were demineralized. They were then divided into two groups at random – Group 1: nHA, Group 2: nFHA – and then into two subgroups (A and B), each of which underwent two distinct in vitro remineralization procedures. The first procedure only used 10-weight percent nHA aqueous slurries, while the second involved first being exposed to fractional CO₂ laser irradiation before nHAp was applied. A micro-Vickers hardness tester and a spectrophotometer were used to quantify microhardness and color, respectively. They came to the conclusion that nHAp has exceptional remineralizing effects on early enamel lesions, effects that are undoubtedly enhanced by the addition of laser therapy.

Amaechi et al. evaluated how two kinds of toothpaste with hydroxyapatite or 500 ppm fluoride promoted remineralization and prevented tooth decay.^[13] In a two-arm, double-blind, randomized crossover study, 30 adults wore intraoral appliances for 14 days each, during which time they were exposed to toothpaste containing either 10% hydroxyapatite or 500 ppm F (amine fluoride), depending on which enamel block had an artificially-produced caries lesion. Lesion depth and baseline and posttest mineral loss were measured using microradiography. They found that 10% hydroxyapatite was as effective as 500 ppm F in remineralizing early caries.

Additional remineralizing and preventive agents are frequently required to enhance caries preventive effect of fluoride in people with high caries risk. The fluoride dose recommended for toddlers and children is even lower than the regulatory 1000–1500 ppm fluoride concentration in nonprescription toothpaste, which is probably suboptimal for effective remineralization of initial lesions. An agent that is as effective as fluoride but whose dosage may be increased without raising any safety issues is anticipated to be a better option, particularly for kids.^[13]

Nourolahian et al. compared the remineralization efficacy of Remin Pro (HAp, fluoride, and xylitol) with CPP-ACP and acidulated phosphate fluoride gel. In their study, 96 enamel samples were prepared. Baseline microhardness was measured for all the samples using the Vickers microhardness test. After developing the initial caries lesions, the microhardness of all the demineralized samples was measured, and the samples were then divided into four groups (n = 24). CPP-ACP in Group 1, Remin Pro in Group 2, and APF gel in Group 3 were placed on the samples for 4 min. The control group received no treatments. The microhardness of the samples was measured again following a pH cycle of 5 days. The data were analyzed by ANOVA and the post hoc test at the significance level of P < 0.05. They reached the conclusion that APF, CPP-ACP, and Remin Pro showed the same remineralization capacity for increasing demineralized enamel hardness.

The present study, along with other studies in the past decade, highlights the efficacy of nonfluoridated agents and dentifrices in caries control. Pediatric and general dentists should strive to learn about such newer remineralizing agents and employ them in their practice.

The limitations of the present study were the need for a larger sample size and a longer observation period after the intervention.

Dentistry in the 21^st century is rapidly changing with innovations in technology and the development of newer dental materials. Given the unique role of the enamel surface layer in the progress of dental caries, the assessment of changes in this region is imperative.^[14] New improved materials capable of enamel remineralization even in low salivary calcium and phosphate ion concentrations are the need of the hour. Some of them are bioactive glass, CPP-ACP, Tri-calcium phosphate, etc.

Considering the results of this in vitro study, it can be assumed that nHAp has a comparable, if not better ability to remineralize primary enamel when compared to CPP-ACP. It was also observed that Er: YAG laser irradiation improved the remineralizing capacity of both the agents used in the study.

Further studies should be conducted in vivo for closer simulation of the oral environment and also by using newer methods of imaging such as FEM and spectrophotometry.